The term “stroke” is a general term that covers conditions having different clinical symptoms. For example, a stroke may be caused by an ischaemic or haemorrhagic insult.
Ischaemic insults (ischaemia) are characterized in a reduction or interruption of the blood circulation in the brain due to a lack of arterial blood supply. Often this is caused by thrombosis of an arteriosclerotic stenosed vessel or by arterio arterial, or cardial embolisms.
Haemorrhagic insults are based inter alia on the perforation of brain supplying arterias damaged by arterial hypertonia. However, only approximately 20% of all cerebral insults are caused by haemorrhagic insults. Thus, stroke due to thrombosis is much more prevalent.
In comparison to other tissue ischaemias, the ischaemia of the neuronal tissue is widely accompanied by necrosis of the effected cells. The higher incidence of necrosis in neuronal tissue can be explained with the new understanding of the phenomenon “excitotoxicity,” which is a complex cascade comprising a plurality of reaction steps. The cascade is initiated by ischaemic neurons affected by a lack of oxygen, which then lose ATP instantaneously and depolarize. This results in an increased postsynaptic release of the neurotransmitter glutamate, which activates membrane bound glutamate receptors regulating cation channels. However, due to the increased glutamate release glutamate receptors become over activated.
Glutamate receptors regulate voltage dependent cation channels, which are opened by a binding of glutamate to the receptor. This results in a Na+ and Ca2+ influx into the cell massively disturbing the Ca2+ dependent cellular metabolism. Especially the activation of the Ca2+ dependent catabolic enzymes result in subsequent cell death (Lee, Jin-Mo et al., “The changing landscape of ischaemic brain injury mechanisms”; Dennis W. Zhol “Glutamate neurotoxicity and diseases of the nervous system”).
Although the mechanism of glutamate mediated neurotoxicity is not yet entirely understood, it is agreed upon that it contributes in a large extent to neuronal cell death following cerebral ischaemia (Jin-Mo Lee, et al.).
The re-opening of a closed vessel has priority in the therapy of acute cerebral ischaemia, in addition to safeguarding vital functions and stabilizing physiological parameters. The re-opening can be performed by different means. The mere mechanical re-opening, as e.g., Percutaneous Transluminal Coronary Angioplasty (PTCA) after heart attack, so far has not yet led to satisfying results. Only with a successful fibrinolysis can an acceptable improvement of the physical condition of patients be achieved. This can be accomplished by a local application using a catheter (PROCAT, a study with pro-urokinase). However, despite initial positive results, this method has not yet been officially approved as a pharmaceutical treatment.
Naturally occurring fibrinolysis is based on the proteolytic activity of the serine protease plasmin, which originates from its inactive precursor by catalysis (activation). The natural activation of plasminogen is catalyzed by the plasminogen activators u-PA (urokinase type plasminogen activator) and t-PA (tissue plasminogen activator) occurring naturally in the body. In contrast to u-PA, t-PA forms a so-called activator complex together with fibrin and plasminogen. Thus, the catalytic activity of t-PA is fibrin dependent and is enhanced in its presence approximately 550-fold. Besides fibrin, fibrinogen can also stimulate t-PA mediated catalysis of plasminogen to plasmin, though to a lesser extent. In the presence of fibrinogen, the t-PA activity only increases 25-fold. Also the cleavage products of fibrin (fibrin degradation products (FDP)) can stimulate t-PA.
Early attempts of thrombolytic treatment of acute stroke go back to the 1950s. Extensive clinical trials with streptokinase, a fibrinolytic agent from beta-haemolysing streptococci, started in 1995. Streptokinase forms a complex with plasminogen that catalyzes other plasminogen molecules into plasmin.
Streptokinase therapy has severe disadvantages since streptokinase is a bacterial protease and therefore can provoke allergic reactions in the body. Furthermore, if a patient had a previous streptococci infection that resulted in a production of antibodies, the patient may exhibit streptokinase resistance, making the therapy more difficult. Besides this, clinical trials in Europe (Multicenter Acute Stroke Trial of Europe (MAST-E), Multicenter Acute Stroke Trial of Italy (MAST-I)) and Australia (Australian Streptokinase Trial (AST)) indicated an increased mortality risk and a higher risk of intracerebral bleeding (intracerebral haemorrhage, ICH) after treating patients with streptokinase. These trials had to be terminated early.
Alternatively, urokinase—also a classical fibrinolytic agent—can be used. In contrast to streptokinase, it does not exhibit antigenic characteristics since it is an enzyme naturally occurring in various body tissues. It is an activator of plasminogen and independent of a co-factor. Urokinase is produced in kidney cell cultures.
Another therapeutic thrombolysis agent tested was a recombinant tissue type plasminogen activator, rt-PA (see EP 0 093 619, U.S. Pat. No. 4,766,075), produced in recombinant hamster cells. In the 1990s several clinical trials were performed world-wide using t-PA with acute myocardial infarction as the main indication, leading to only partially understood and contradictory results. In the European Acute Stroke Trial (ECASS) patients were treated within a time frame of 6 hours after the onset of the symptoms of a stroke intravenously with rt-PA. After 90 days the mortality rate as well as the Barthel-Index were examined as an Index for the disability or the independent viability of patients. No significant improvement of the viability was reported but an increase of mortality was seen. Thus, a thrombolytic treatment with rt-PA of patients being individually selected according to their respective case history immediately after the beginning of the stroke could possibly be advantageous. However, a general use of rt-PA within the time frame of 6 hours after the onset of stroke was not recommended since an application during this time increased the risk of intracerebral haemorrhage (ICH) (C. Lewandowski C and Wiliam Barsan, 2001: Treatment of Acute Stroke; in: Annals of Emergency Medicine 37:2; S. 202 ff.).
The thrombolytic treatment of stroke was also subject of a clinical trial conducted by the National Institute of Neurologic Disorder and Stroke (so called NINDS rtPA Stroke Trial) in the USA. This trial concentrated on the effect of intravenous rt-PA treatment within only three hours after the onset of the symptoms. Patients were examined three months after the treatment. Due to the observed positive effects of this treatment on the viability of patients rt-PA treatment-within the limited time frame of three hours was recommended, although the authors found a higher risk for ICH.
Two further studies (ECASS II Trial: Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischaemic Stroke (ATLANTIS)) examined whether the positive effects of rt-PA treatment within three hours after the onset of stroke could be repeated instead with a treatment within six hours time. However, the results indicated that there was no improvement in the clinical symptoms, nor was there any decrease in mortality. Further, the higher risk for ICH remained.
These partially contradictory results led to a high caution in the use of rt-PA. A 1996 publication of the American Heart Association pointed out the strong skepticism among doctors with respect to thrombolytic treatment of stroke; in contrast, there is no such skepticism with respect to fibrinolytica in the therapy of myocardical infarct (van Gijn J, MD, FRCP, 1996-Circulation 1996, 93: 1616-1617).
A rationale behind this skepticism was first given in a summary of all stroke trials published 1997 (updated in March 2001). According to this review all thrombolytica treatments (urokinase, streptokinase, rt-PA or recombinant urokinase) resulted in a significant higher mortality within the first 10 days after a stroke, while the total number of either dead or disabled patients was reduced when the thrombolytica were applied within six hours after stroke onset. This effect was mainly due to ICH. Such results gave reason to some to make the sarcastic statement that stroke patients had the choice to either die or to survive disabled (SCRIP 1997: 2265, 26). The broad use of thrombolytica for the treatment of stroke was therefore not recommended.
Nevertheless, the therapy with rt-PA currently is the only treatment of acute cerebral ischaemia approved by the Food and Drug Administration (FDA) in the USA. However, it is restricted to administration within three hours after the onset of stroke.
The approval of rt-PA was reached in 1996. In 1995, first announcements about negative side effects of t-PA became known, which provide an explanatory basis for its dramatic effects when applied in stroke treatment outside the three hour time frame. Microglia cells and neuronal cells of the hippocampus produce t-PA, which contributes to the glutamate mediated excitotoxicity. This was concluded from a comparative study on t-PA deficient and wild type mice where glutamate agonists were injected in their hippocampuses. The t-PA deficient mice showed a significant higher resistance against external (inthrathecal) applicated glutamate (Tsirka S E et al., Nature, Vol. 377, 1995, “Excitoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator”). These results were confirmed in 1998, when Wang et al. demonstrated a nearly double quantity of necrotic neuronal tissue in t-PA deficient mice when t-PA was injected intravenously. This negative effect of external t-PA on wild type mice was only approximately 33% (Wang et al., 1998, Nature, “Tissue plasminogen activator (t-PA) increases neuronal damage after focal cerebral ischaemia in wild type and t-PA deficient mice”.)
Further results on the stimulation of excitotoxicity by t-PA were published by Nicole et al. in the beginning of 2001 (Nicole O., Docagne F Ali C; Margaill I; Carmeliet P; MacKenzie E T, Vivien D and Buisson A, 2001: The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling; in: Nat Med 7, 59-64). They showed that t-PA being released by depolarized cortical neurons could interact with the NR1 sub-unit of the glutamate receptor of the NMDA type, leading to a cleavage of NR1. This increased the receptor's activity, resulting in greater tissue damage after glutamate agonist NMDA was applied. The NMDA agonist induced excitotoxicity. Thus, t-PA exhibits a neurotoxic effect by activating the glutamate receptor of the NMDA type.
According to a further explanation, the neurotoxicity of t-PA results indirectly from the conversion of plasminogen in plasmin. According to this model plasmin is the effector of neurotoxicity (Chen Z L and Strickland S, 1997: Neuronal Death in the hippocampus is promoted by plasmin-catalysed degradation of laminin. Cell: 91, 917-925).
An outline summarizing the time depending neurotoxic effect of t-PA is given in FIG. 5. The increased toxicity of the recombinant t-PA compared to endogenic t-PA is also evident in FIG. 5. This increased toxicity is probably due to rt-PA being able to enter into tissue in higher concentrations.
Despite its neurotoxic side effect and its increasing effect on mortality, t-PA was approved by FDA. This can only be explained by the lack of harmless and effective alternatives. Therefore, there is still a need for safe therapies. However, if they were still based on thrombolytica, in case it is not possible to find alternatives to thrombolysis, the problem of neurotoxicity has to be considered (see for example Wang et al. a.a.O.; Lewandowski and Barson 2001 a.a.O.).
Further examination of known thrombolytica including DSPA (Desmodus rotundus Plasminogen Activator) as possible new drug candidates for stroke was terminated, even though principally all thrombolytica were potentially suitable. The potential suitability of DSPA for this medical indication was pointed out earlier (Medan P; Tatlisumak T; Takano K; Carano R A D; Hadley S J; Fisher M: Thrombolysis with recombinant Desmodus saliva plasminogen activator (rDSPA) in a rat embolic stroke model; in: Cerebrovasc Dis 1996:6; 175-194 (4th International Symposium on Thrombolic Therapy in Acute Ischaemic Stroke). DSPA is a plasminogen activator with a high homology (resemblance) to t-PA. Therefore—and in addition to the disillusionment resulting from the neurotoxic side effects of t-PA—there were no further expectations for DSPA being a suitable drug for stroke treatment.
Instead, recent strategies aiming to improve known thrombolytic treatments tried to apply the thrombolytic substance intraarterially, rather than intravenously via a catheter positioned close to the intravascular thrombus. The initial experiments were performed with recombinant produced urokinase. The hope was that the necessary dose for thrombolysis could be reduced and that the negative side effects could be reduced. However, this application requires a high technical expenditure and is not available everywhere. Furthermore, the patient has to be prepared in a time consuming action. Time, however, is often limited when treating for stroke. Thus, the additional preparation time adds an additional risk.
Presently, new concepts are directed to anticoagulants such as heparin, aspirin or ancrod, which is the active substance in the poison of the malayan pit viper. Two clinical trials examining the effects of heparin (International Stroke Trial (IST) and Trial of ORG 10172 in Acute Stroke Treatment (TOAST)) however, did not indicate a significant improvement of mortality or a prevention of stroke.
A further new treatment did not focus on thrombus, blood thinning or anti coagulation, but instead attempted to increase the vitality of cells damaged by the interruption of blood supply (WO 01/51613 A1 and WO 01/51614 A1). To achieve this, antibiotics from the group of quinons, aminoglycosides or chloramphenicol were applied. In an alternative strategy, a research group administered citicholin immediately after the onset of stroke. In the body, citicholin is cleaved to cytidine and choline. The cleavage products form part of the neuronal cell membrane and thus support the regeneration of damaged tissue (U.S. Pat. No. 5,827,832).
Recent research on safe treatment is based on the new finding that a part of the fatal consequences of stroke is caused only indirectly by interrupted blood supply but directly to excito- or neurotoxicity, including over-activated glutamate receptors. This effect is increased by t-PA (see above). A strategy to reduce excitotoxicity would be, therefore, to administer neuroprotectives. Neuroprotectives can be used separately or in combination with fibrinolytic agents in order to minimize neurotoxic effects. They can lead to a reduced excitotoxicity either directly, e.g. as a glutamate receptor antagonist, or indirectly, e.g., by inhibiting voltage dependent sodium or calcium channels (Jin-Mo Lee et al. a.a.O.).
A competitive inhibition (antagonistic action) of the glutamate receptor of NMDA type can be achieved, e.g., with 2-amino-5-phosphonovalerate (APV) or 2-amino-5-phosphonoheptanoate (APH). A non competitive inhibition can be achieved, e.g., by substances binding to the phencyclidine side of the channels. Such substances can be phencyclidine, MK-801, dextrorphane or cetamine.
So far, treatments with neuroprotectives have not shown the expected success, possibly because neuroprotectives had to be combined with thrombolytic agents in order to exhibit their protective effects. This also applies to other substances (see also FIG. 6).
Even a combination of t-PA and neuroprotective agents results only in limited damage. Nevertheless, the disadvantageous neurotoxicity of the fibrinolytic agent as such is not avoided.